A long-term objective of this proposal is to define the molecular details and mechanisms of receptor mediated endocytosis and receptor reutilization. Endocytosis of many hormones and growth factors may play an important role in the ability of such regulatory molecules to control cellular metabolism and growth. It is presently not known how the internalization of regulatory molecules by the cell is related to the cell's ability to respond to those molecules. Elucidation of how cells normally receive the appropirate signals from these molecules should lead to a better understanding of diseases (such as some forms of diabetes mellitus or cancer) in which the regulation of metabolism or growth control is abnormal. We have chosen to study the rat hepatic asialoglycoprotein (or galactosyl) receptor because this system offers an excellent opportunity to determine the details, at both the cellular and molecular levels, of receptor function during endocytosis. The asialoglycoprotein receptor will bind a variety of experimentally useful ligands and has been shown to be reutilized or recycled both in vitro and in vivo. The receptor has been purfied and characterized and has also been reconstituted and studied in synthetic lipid membranes. We propose to answer several currently important questions about endocytosis and recycling of the asialoglycoprotein receptor, including whether receptor-ligand complexes enter the cell and, if so, what the intracellular pathways of the receptor and ligand are. We will also obtain such information as when and where the ligand and receptor separate, whether other cellular components interact with the receptor and how the receptor is reutilized or recycled. Radioactive, fluorescent, electron dense and photoaffinity derivatives specific for the receptor will be used to monitor the cellular location and function of the receptor during endocytosis and recycling. Organic synthetic chemistry, fluorescence and video intensification microscopy, electron microscopy and general biochemical and cell biology techniques will be used in these studies.